Dengue virus (DENV) infections result in an estimated 50 million cases of self-limited Dengue Fever (DF) every year and 250,000 to 500,000 cases of life-threatening Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS), which is characterized by thrombocytopenia, coagulopathy, complement depletion, and increased vascular permeability with leakage of plasma from the vascular system. The pathogenesis of DF and DHF/DSS is complex and poorly understood, but DENV NS1 protein, which is secreted by infected cells and found in infected patient sera, is likely to play an important role. NS1 can directly activate complement, and levels of soluble NS1 (sNS1) in patient sera correlate with disease severity. Furthermore, multiple studies indicate that antibodies to NS1 can be immunopathogenic. We hypothesize that DENV sNS1 and anti-NS1 antibodies contribute to DHF/DSS by modulating viral replication, activating complement, triggering thrombocytopenia and coagulopathy, and increasing vascular permeability. The role of sNS1 in viral infection and the mechanisms by which sNSI and anti-NS1 antibody contribute to clinical features of dengue disease have been difficult to study due to the lack of an appropriate mouse model. We have developed new mouse models that include the use of non-neuroadapted virus administered via relevant routes and at reasonable doses to produce peripheral tissue tropism and, at higher doses, fatal plasma leakage. These mouse models will be used to achieve the following specific aims: Specific Aim 1: Characterize hematologic disturbances induced by sNSI and anti-NS1 antibodies. Aim 1A: Characterize serotype cross-reactivity and platelet-binding activities of anti-NS1 antibodies. Aim 1B: Measure the effects of sNS1 and anti-NS1 antibodies on platelets, coagulation, and vascular permeability in mice. Aim 1C: Determine the role of complement activation in sNS1- and anti-NS1 antibody-induced phenotypes. Specific Aim 2: Evaluate the effects of sNS1 and anti-NS1 antibodies on DENV pathogenesis in mice. Aim 2A: Assess effects of sNS1 and anti-NS1 antibody on DENV replication and thrombocytopenia. Aim 2B: Assess the effects of sNS1 and anti-NS1 antibody on DENV-induced vascular permeability. This research will produce information critical to the development of a safe and effective dengue vaccine, a global public health priority. NS1 is a proposed target of dengue vaccines, and most vaccine candidates currently in clinical trials include NS1. It is critical to determine if the induced antibody responses will be protective, or alternatively, deleterious to patient health. [unreadable] [unreadable] [unreadable]